The goal of this proposal is to use genomic approaches to identify new therapeutic targets for head and neck squamous cell carcinoma (HNSCC), by defining the p63 pathway in tumors in vivo. The p53 family member p63 is overexpressed in a majority of primary HNSCC tumors and is essential for epithelial precursor development in vivo, suggesting a role for this gene in tumorigenesis. Our recent data using small inhibitory RNA (siRNA) targeted against specific p63 isoforms demonstrates that loss of p63 expression induces apoptosis in HNSCC cells that overexpress p63. In contrast, p63 inhibition has no effect on survival of normal keratinocytes or of tumor cells that do not express p63. To date, the downstream mechanisms of p63 remain poorly characterized. Therefore it will first be determined whether death following p63 inhibition depends on intact function of p53 or the related gene p73, and which p63 isoform(s) provide the survival effect. Second, the downstream genes that mediate tumor-associated effects of p63 will be identified using expression profiling following p63-specific siRNA in HNSCC cells. To confirm putative p63 targets in vivo, microdissected primary tumors from the MGH/MEEI tumor bank will be used to correlate p63 levels with genome-wide expression profiles in primary HNSCC specimens. Comparison of genes regulated following p63 siRNA to genes expressed coincident with p63 in primary tumors should allow identification of the most biologically relevant p63 targets. Third, the potential therapeutic relevance of the confirmed p63 targets will be tested directly in HNSCC using an siRNA approach. Together these studies will define the relevance of the p63 pathway in tumorigenesis, and will identify potential new therapeutic targets that mediate the critical survival effect of p63 in HNSCC. [unreadable] [unreadable]